The invention relates to antiviral and virucidal compositions and methods and to methods of incorporating a polypeptide into a virus or a virus vector.
Human immunodeficiency virus type 1 (HIV-1), a lentivirus, exhibits a complex viral life cycle. Replication of HIV-1 is tightly regulated by numerous cell-and virus-encoded regulatory proteins (Cullen, 1992. Microbiol. Rev. 56:375-394). Essential virus-encoded enzymes, including reverse transcriptase (RT), ribonuclease H (RNaseH), protease (PR), and integrase (IN), do not have cell-encoded counterparts, and for this reason have been used as targets for developing agents which inhibit virus replication without significantly adversely affecting cells (Debouck, 1992, AIDS Res. Hum. Retrovir. 8:153-164; Ridky et al., 1995, J. Biol. Chem. 270:29621-29623; Miller et al., 1996, AIDS Res. Hum. Retrovir. 12:859-865). Although considerable advances have been made in developing agents effective for inhibiting RT and PR, the mutability of HIV-1 has led to strains of the virus which exhibit resistance to such agents (Richman, 1995, Clin. Infect. Dis. 21 (Suppl. 2):S166-S169). Thus, there is a pressing need to develop alternative anti-HIV therapeutic strategies.
Specific cell compartmental localization of therapeutic moieties influences their therapeutic effect. For example, single chain variable region antibody fragments (SFvs) which bind specifically to HIV-1 IN and which are localized in the cytoplasm of a T lymphocyte inhibit infection of the lymphocyte more efficaciously than SFvs which are concentrated in the nucleus of the lymphocyte (Levy-Mintz et al., 1996, J. Virol. 70:8821-8832). Ribozymes have been localized within the virion of murine leukemia virus (MuLV; Rothman et al., 1996, Science 272:227-234). Incorporation of chimeric proteins into retrovirus particles has been reported, wherein the chimeric proteins had amino acid sequences which comprised a portion of the amino acid sequence of a non-viral protein and a portion of either HIV-1 Gag protein or HIV-1 Vpr protein (Wu et al., 1995, J. Virol. 69:3389-3398; Wu et al., 1996, Virology 219:307-313; Jones et al., 1990, J. Virol. 64:2265-2279; Wang et al., 1994, Virology 200:524-534; Weldon et al., 1990, J. Virol. 64:4169-4179).
It has been suggested that sorting of most cellular proteins into specific compartments is determined by protein-protein interactions mediated by specific domains of the proteins involved (Rothman et al., supra). This model of protein sorting is designated the protein-docking model. For example, interaction between a domain of a membrane protein with the signal sequence domain of a cytoplasmic protein can result in export of the cytoplasmic protein into either the Golgi apparatus or a mitochondrion (Rothman et al., supra).
Numerous proteins are packaged into HIV-1 virions, including RT, RNaseH, PR, IN, and proteins designated Gag, Pol, and Env. The genome of HIV-1 encodes other proteins which are packaged into HIV-1 virions, including a protein designated Vpr, which is present in virions of all primate lentivirus (Tristem et al., 1992, EMBO J. 11:3405-3412). The role of Vpr in infection of cells by lentiviruses has been studied extensively. Vpr is expressed relatively late in the lentiviral life cycle and encodes a 14 kilodalton protein which is predominantly localized in the nucleus of an infected cell (Wong-Staal et al., 1987, AIDS Res. Hum. Retrovir. 3:33-39; Lu et al., 1993, J. Virol. 67:6542-6550). Vpr is reported to be incorporated into lentivirus particles in quantities equal to the quantity of Gag protein (Lu et al., Id.; Cohen et al., 1990, J. Virol. 64:3097-3099).
Prior art investigations indicate that the carboxyl terminal domain (p6 region) of the Gag precursor designated p55 is involved in packaging of Vpr into HIV-1 virions (Lavallee et al., 1994, J. Virol. 68:1926-1934; Kondo et al., 1996, J. Virol. 70:159-164; Lu et al., 1995, J. Virol. 69:6873-6879; Paxton et al., 1993, J. Virol. 67:7229-7237). None of these reports identified a site at which p55 and Vpr interact within the amino acid sequence of either Vpr or the p6 region. A direct interaction between Vpr and the nucleocapsid protein designated Ncp7 has been demonstrated (Lim Tung et al., 1997, FEBS Lett. 401:197-201; De Rocquigny et al., 1997, J. Biol. Chem., J. Biol. Chem. 272:30753-30759). This interaction is mediated in vitro by the zinc finger regions of Ncp7 and the sixteen carboxyl terminal amino acids of Vpr (De Rocquigny et al., Id.). It may be that binding of Ncp7 in cooperation with another HIV-1 protein, possibly the p6 region of Gag, induces incorporation of Vpr into mature HIV-1 particles (De Rocquigny et al., Id.).
Several biological functions of Vpr have been defined. For example, Vpr is able to transactivate several heterologous viral promoters which do not share a common DNA sequence element (Cohen et al., 1990, J. Acquir. Immune Defic. Syndr. 3:11-18). Vpr is essential for optimal infection of macrophages by HIV-1 and influences nuclear transport of the HIV-1 pre-integration complex (Balliet et al., 1994, Virology 200:623-631; Connor et al., 1995, Virology 206:935-944; Westervelt et al., 1992, J. Virol. 66:3925-3931; Heinzinger et al., 1994, Proc. Natl. Acad. Sci. USA 91:7311-7315). Vpr also activates transcription from the HIV-1 long terminal repeat (LTR), and influences terminal differentiation of certain cell-types, such as rhabdomyosarcoma cells (Agostini et al., 1996, J. Mol. Biol. 261:599-606; Cohen et al., 1990, J. Acquir. Immune Defic. Syndr. 3:11-18; Wang et al., 1995, J. Biol. Chem. 270:25564-25569; Levy et al., 1993, Cell 72:541-550). Addition of exogenous Vpr to cells latently infected with HIV-1 can reactivate replication of the virus, indicating that Vpr may increase HIV-1 expression by affecting transcriptional or translational events (Levy et al., 1994, Proc. Natl. Acad. Sci. USA 91:10873-10877; Levy et al., 1995, J. Virol. 69:1243-1252). Vpr also causes cell cycle arrest in the G2/M phase and is capable of inducing apoptosis following cell cycle arrest (He et al., 1995, J. Virol. 69:6705-6711; Jowett et al., 1995, J. Virol. 69:6304-6313; Re et al., 1995, J. Virol. 69:6859-6864; Rogel et al., 1995, J. Virol. 69:882-888; Stewart et al., 1997, J. Virol. 71:5579-5592). All of these effects are probably mediated by interactions between Vpr and one or more cellular proteins.
Vpr is able to associate with the major uracil DNA glycosylase (UDG) involved in cellular DNA repair (Slupphaug et al., 1995, Biochemistry 34:128-138; BouHamdan et al., 1996, J. Virol. 70:697-704). The cellular physiological role of UDGs and deoxyuracil triphosphate pyrophosphatases (dUTPases) is believed to be prevention of misincorporation of deoxyuracil into DNA during DNA synthesis. A recent report excludes involvement of UDG in contributing to G2 arrest of cells. Mutational analysis of Vpr has been used to demonstrate that binding of Vpr to UDG is neither necessary nor sufficient to effect cell cycle arrest (Selig et al., 1997, J. Virol. 71:4842-4846). It may be that association of Vpr with UDG permits incorporation of UDG into HIV-1 virions, with the result that, upon subsequent infection of a cell by such a virion, uracil mis-incorporation into DNA transcribed from HIV-1 RNA is reduced. Thus, UDG encoded by a host cell genome may have a physiological role in the infectious cycle of HIV-1 that is similar to the role of dUTPases of non-primate lentiviruses. It has been reported that a strain of caprine arthritis encephalitis virus which is deficient in dUTPase accumulates G-to-A substitutions in its genome in vivo (Turelli et al., 1997, J. Virol. 71:4522-4530). It has also been reported that the vpr gene partially accounts for the lower-than-predicted in vivo mutation rate of HIV-1 (Mansky, 1996, Virology 222:391-400).
The ability of Vpr to associate with other cellular proteins, including glucocorticoid receptors, the basal transcription factor TFIIB, transcription factor Sp1, and the cellular DNA repair protein designated HH23A has been reported (Refaeli et al., 1995, Proc. Natl. Acad. Sci. USA 92:3621-3625; Agostini et al., 1996, J. Mol. Biol. 261:599-606; Wang et al., 1995, J. Biol. Chem. 270:25564-25569; Withers-Ward et al., 1997, J. Virol. 71:9732-9742). It has also been reported that the portion of Vpr that interacts specifically with TFIIB is located between or including amino acid residues 15 and 77 of Vpr (Agostini et al., 1996, J. Mol. Biol. 261:599-606), and that the amino terminal domain of TFIIB is involved in this interaction. At least a portion of the region of HH23A comprising the forty-five carboxyl terminal amino acids of that protein interacts with Vpr. Despite identification of several proteins which specifically interact with Vpr, the mechanism(s) which mediate such interactions are not understood.
It has been suggested that virions derived from a retrovirus such as HIV-1 may be useful as vectors for delivery of a nucleic acid to cells of an animal such as a human (Miller et al., 1989, BioTechniques 7:980-982; Cometta et al., 1993, Hum. Gene Ther. 4:579-588; Salmons et al., 1993, Hum. Gene Ther. 4:129-141). A significant difficulty in designing retrovirus-derived vectors relates to packaging a desired protein into a vector virion.
Because the Vpr protein can be packaged into HIV-1 virions in an amount analogous to the amounts of the major HIV-1 structural proteins, it has been suggested that chimeric proteins having an amino acid sequence comprising a portion of Vpr can be incorporated into HIV-1 -derived virions (Lu et al., 1993, J. Virol. 67:6542-6550; Cohen et al., 1990, J. Virol. 64:3097-3099). Incorporation of a chimeric protein into an HIV-1-derived virion has been reported using a chimeric protein having an amino acid sequence comprising a portion of the sequence of a non-HIV protein and a portion of the sequence of Vpr (Wu et al., 1995, J. Virol. 69:3389-3398; Wu et al., 1996, Virology 219:307-313). For example, a chimeric protein having an amino acid sequence comprising a portion of the sequence of chloramphenicol acetyl-transferase (CAT) and a portion of the sequence of Vpr has been incorporated into an HIV-1-derived virion, and the virion-associated CAT chimeric protein retained CAT activity (Id.). Also, for example, chimeric proteins have been made, each having an amino acid sequence comprising a portion of the amino acid sequence of either HIV-1 Vpr or HIV-2 Vpx and a portion of the amino acid of a protein selected from the group consisting of CAT, staphylococcal nuclease (SN), wild type PR, mutated PR, wild type IN, mutated IN, wild type RT, and mutated RT (Wu et al., 1995, J. Virol. 69:3389-3398; Wu et al., 1996, Virology 219:307-313; Liu et al., 1997, J. Virol. 71:7704-7710; Wu et al., 1997, EMBO J. 16:5113-5122).
The situations described above have severe drawbacks. For example, in certain circumstances, chimeric proteins having an amino acid sequence comprising a portion of the amino acid sequence of Vpr and a portion of the amino acid sequence of a protein other than Vpr may not be suitable for use as anti-viral therapeutic agents. Vpr can arrest the cell cycle when it is present in a cell, and can also induce cellular apoptosis. Such chimeric proteins might also reactivate viral replication in a patient harboring a latent retrovirus, and competition between such a chimeric protein and wild-type Vpr may permit viral escape, meaning that the virus can survive drug treatment and continue to replicate in the individual. Furthermore, such fusion proteins may not exhibit the activity normally associated with the non-Vpr protein component of the chimeric protein.
Thus, the methods of the prior art for incorporating a chimeric protein into an HIV-1 virion have several serious limitations. The present invention overcomes these limitations, in that the present invention does not require use of a chimeric protein having an amino acid sequence comprising a portion of the amino acid sequence of Vpr to incorporate a protein into a virion.
The invention relates to a polypeptide comprising a Vpr-binding region having the amino acid sequence
Xaa1-Xaa2-Xaa3-Phe (SEQ ID NO: 30). wherein Xaa1 is selected from the group consisting of Trp and Phe, wherein each of Xaa2 and Xaa3 is any amino acid residue, and wherein the polypeptide does not normally comprise the region. In one aspect, Xaa1 is Trp. In another aspect, Xaa2 is selected from the group consisting of Ala, Trp, His, Phe, and Tyr. In yet another aspect, Xaa3 is selected from the group consisting of Gln, Thr, Ala, His, Ser, Asp, Glu, and Phe. In still another aspect, Xaa2 is selected from the group consisting of Ala, Trp, His, Phe, and Tyr. In another aspect, Xaa2 is Trp.
In one embodiment of the polypeptide of the invention, the polypeptide comprises a plurality of the Vpr-binding regions, wherein for each of the regions, Xaa1 is independently selected from the group consisting of Trp and Phe, and each of Xaa2 and Xaa3 is independently any amino acid residue. In one aspect, a linker region comprising at least about four amino acid residues is interposed between two of the Vpr-binding regions. In another aspect, the linker region has the amino acid sequence
Gly-Gly-Gly-Cys (SEQ ID NO: 33).
In still another aspect, the polypeptide has an amino acid sequence comprising the sequence
Trp-aa2-Xaa3-Phe-Gly-Gly-Gly-Cys-Trp-Xaa5-Xaa6-Phe (SEQ ID NO: 34),
wherein each of Xaa2, Xaa3, Xaa5, and Xaa6 is any amino acid residue.
The invention also relates to an isolated nucleic acid encoding the polypeptide of the invention, and to a cell comprising that isolated nucleic acid.
The invention further relates to a virion comprising an isolated nucleic acid encoding the polypeptide of the invention. In one aspect, the primate lentivirus is selected from the group consisting of a virion of a virus vector and a primate lentivirus, such as HIV-1.
The invention still further relates to the polypeptide of the invention in substantially pure form.
The invention also relates to a polypeptide comprising a Vpr-binding region, wherein the polypeptide does not normally comprise the region, and wherein the region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-29. In one aspect the region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-22.
The invention still further relates to a method of rendering a polypeptide capable of binding with Vpr and to a polypeptide made by this method. The method comprises altering the polypeptide such that the altered polypeptide comprises a Vpr-binding region having the amino acid sequence
Xaa1-Xaa2-Xaa3-Phe (SEQ ID NO: 30),
wherein Xaa1 is selected from the group consisting of Trp and Phe, wherein each of Xaa2 and Xaa3 is any amino acid residue, and wherein the polypeptide does not normally comprise the region. When the polypeptide is altered such that it comprises the region, the altered polypeptide is capable of binding with Vpr. In one aspect of this method, the altered polypeptide comprises a plurality of Vpr-binding regions. For each region Xaa1 is independently selected from the group consisting of Trp and Phe, and each of Xaa2 and Xaa3 is independently any amino acid residue. In another aspect, the linker region has the amino acid sequence
Gly-Gly-Gly-Cys (SEQ ID NO: 33).
In still another aspect, the polypeptide has an amino acid sequence comprising the sequence
Trp-Xaa2-Xaa3-Phe-Gly-Gly-Gly-Cys-Trp-Xaa5-Xaa6-Phe (SEQ ID NO: 34),
wherein each of Xaa2, Xaa3, Xaa5, and Xaa6 is any amino acid residue. The polypeptide may, for example, be an antiviral agent, a virucidal agent, a cellular therapeutic polypeptide, or a cellular suppressant polypeptide.
The invention also relates to a method of rendering a polypeptide susceptible to incorporation into a virion of a virus which normally expresses Vpr and to a polypeptide made by this method. This method comprises altering the polypeptide such that the polypeptide comprises a Vpr-binding region having the amino acid sequence
Xaa1-Xaa2-Xaa3-Phe (SEQ ID NO: 30),
wherein Xaa1 is selected from the group consisting of Trp and Phe, wherein each of Xaa2 and Xaa3 is any amino acid residue, and wherein the polypeptide does not normally comprise the region. When the polypeptide comprises the region, the polypeptide is susceptible to incorporation into the virion. In one aspect, the virus is a virus vector or a primate lentivirus, such as HIV-1.
The invention further relates to a method of generating a virion comprising a portion of a first polypeptide which does not normally comprise a Vpr-binding region. This method comprises a) providing to a cell a nucleic acid which encodes a second polypeptide having an amino acid sequence which comprises the sequence of the portion and the sequence
Xaa1-Xaa2-Xaa3-Phe (SEQ ID NO: 30),
wherein Xaa1 is selected from the group consisting of Trp and Phe, and wherein each of Xaa2 and Xaa3 is any amino acid residue, b) providing a competent portion of the genome of a virus which normally expresses Vpr to the cell; and c) thereafter incubating the cell under conditions such that the nucleic acid and the competent portion are expressed, whereby the virion comprising the portion of the first polypeptide is generated. In one aspect the portion comprises every amino acid residue of the first polypeptide.
The invention also relates to a method of providing a polypeptide which does not normally comprise a Vpr-binding region to a human cell. This method first comprises generating a virion by a) providing to a cell a nucleic acid which encodes a second polypeptide having an amino acid sequence which comprises the sequence of the portion and the sequence
Xaa1-Xaa2-Xaa3-Phe (SEQ ID NO: 30),
wherein Xaa1 is selected from the group consisting of Trp and Phe, and wherein each of Xaa2 and Xaa3 is any amino acid residue, b) providing a competent portion of the genome of a virus which normally expresses Vpr to the cell; and c) thereafter incubating the cell under conditions such that the nucleic acid and the competent portion are expressed, whereby the virion comprising the portion of the first polypeptide is generated. This method further comprises contacting the virion with the human cell. The polypeptide is provided to the human cell when the virion enters the human cell.
The invention also relates to a method of inhibiting replication in a cell of a human patient of a virus which normally expresses Vpr. This method comprises providing to the cell a polypeptide comprising a Vpr-binding region, wherein the region has the amino acid sequence
Xaa1-Xaa2-Xaa3-Phe (SEQ ID NO: 30),
wherein Xaa1 is selected from the group consisting of Trp and Phe, wherein each of Xaa2 and Xaa3 is any amino acid residue, and wherein the polypeptide does not normally comprise the region. Incorporation of the polypeptide into a virion of the virus is detrimental to replication of the virus. In one aspect of this method, the virion is HIV-1.
The invention further relates to a method of determining whether a polypeptide comprises a Vpr-binding region. The method comprises a) contacting a suspension comprising the polypeptide with at least a portion of Vpr connected to a first support, b) separating the first support from the suspension, and c) assessing whether the polypeptide is associated with the first support. If the polypeptide is associated with the first support then the polypeptide comprises a Vpr-binding region. In one aspect of this method, the portion of Vpr is either His-tagged Vpr or a GST-Vpr chimeric protein. When the portion of Vpr is a GST-Vpr chimeric protein, one aspect of the method further comprises contacting the suspension with a second support prior to contacting the suspension with the chimeric protein. The second support has GST attached thereto. In another embodiment of this aspect, each of the first and second supports comprise a glutathione residue and the polypeptide is a chimeric protein having an amino acid sequence identical to that of a portion of a phage coat protein or a random amino acid sequence.